For LSCM analysis, cells adhered on coverslips from T0 and T4 of both conditions were fixed with 4% formaldehyde in seawater for 1 hour and incubated with 10 μg/ml Nile Red (Sigma-Aldrich, St. Louis, Missouri, USA) for 30 minutes, then washed twice in PBS (pH7.4). The coverslips were then mounted on slides with FluoroshieldTM with DAPI (Sigma-Aldrich) and taken to a TCS SPE microscope (Leica Microsystems, Wetzlar, Germany). The excitation wavelengths were 405 nm and 488 nm and fluorescence emission peaks were: 440 nm (DAPI), 535 nm (Nile Red) and 650 nm (Chlorophyll a). Images acquisition resulting resolution was 2,048 x 2,048 and, to improve the image quality, they were processed by 3D deconvolution with LAS AF software (Leica Microsystems Company).
Tcs spe microscope
Manufactured by Leica Microsystems
Sourced in Germany
The TCS SPE microscope is a confocal laser scanning microscope designed for high-resolution imaging. It features a compact and modular design, allowing for flexible configuration to meet the needs of various applications. The microscope provides advanced optical performance and a range of imaging capabilities to support research and analysis in diverse scientific fields.
Automatically generated - may contain errors
Lab products found in correlation
Las af lite, by Leica camera (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Aqua poly mount, by Polysciences (2 mentions)
Fluoroshieldtm with dapi, by Merck Group (1 mentions)
Nile red, by Merck Group (1 mentions)
Apochromat na 1.3 oil objective lens, by Leica Microsystems (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
7 protocols using tcs spe microscope
1
Lipid Analysis of Cells by LSCM
2
Confocal Microscopy Imaging Protocol
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Confocal images were acquired at RT with a TCS SPE microscope from Leica Microsystems equipped with a Plan Fluor 10 × dry objective lens NA = 0.30, 40 × Apochromat NA = 1.15 oil objective lens, and 63 × Apochromat NA = 1.3 oil objective lens (Leica Microsystems GmbH) and 4 different lasers lines (405, 488, 565, and 647 nm). Pictures were acquired using the Leica software LAS AF v2.2.1 software (Leica Microsystems GmbH) and representative slices were converted to TIFF files using ImageJ software. Transmitted light images were acquired using a microscope (DM 1000, Leica) with a DFC450 CCD camera (Leica Microsystems GmbH) using Leica Application Suite (v4.1). Sections were photographed with Plan 4 × dry objective lens (NA = 0.1) and insets with Plan S-Fluor 20 × or 40 × dry objective lens (NA = 0.90, Nikon) at RT.
3
Immunocytochemistry of N2a Cells
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N2a cells cultured on coverslips placed in 35 mm dishes (30,000 cels/cm2 (link)) were fixed in 4% PFA for 15 min. After washing with PBS, cells were permeabilized with 0.1% Triton X-100 and blocked with 5% goat serum and 10% FBS for 1 h at room temperature. After washing with 3% BSA in PBS, cells were incubated for 1 h with primary antibodies against P2X7R (1:200), Sp1 (1:200) and/or α-tubulin (1:500). Subsequently, cells were washed with PBS and incubated for 1 h with appropriate secondary Alexa Fluor® conjugate antibodies (1:400) and nuclei were counterstained with DAPI. Coverslips were mounted using Prolong® gold antifade reagent (Molecular Probes). Confocal images were acquired with a TCS SPE microscope from Leica Microsystems with a 63× Apochromat NA = 1.3 oil objective lens (Wetzlar, Germany) and analyzed with ImageJ software.
4
Immunofluorescence Staining of Cultured Cells
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Cells plated onto coverslips were stimulated as described previously, washed with PBS, and fixed in 4% paraformaldehyde for 15 min at room temperature. After washing three times with PBS, the cells were permeabilized with 0.2% Triton X-100 and blocked with 2% BSA in PBS for 1 h at room temperature. The cells were then incubated overnight at 4ºC with the primary antibodies anti-MKP1, anti-pERK or anti-pp38 (1:100), and anti-GFAP (1:500). Subsequently, the cells were washed with PBS and incubated with FITC (1:200) or Cy3 (1:400) conjugated secondary antibodies for 1 h at room temperature. The nuclei were counterstained with DAPI (Invitrogen, Barcelona, Spain), and the coverslips were mounted onto glass slides using Aqua-Poly/Mount (Polysciences Europe GmbH, Bergstrasse, Germany). Confocal images were acquired on a TCS SPE microscope from Leica Microsystems (Wetzlar, Germany), and they were analyzed using the ImageJ and Leica LAS AF Lite software.
5
Immunofluorescence Staining of Cultured Cells
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Cells plated on coverslips were stimulated as described previously, washed with PBS and fixed in 4% paraformaldehyde for 15 min at room temperature. After washing three times with PBS, the cells were permeabilized with 0.2% Triton X-100 and blocked with 2% BSA in PBS for 1 h at room temperature. The cells were then incubated overnight at 4°C with primary antibodies against either MKP3 or pERK (1:100), anti-βIII tubulin (1:1000) and anti-GFAP (1:500). Subsequently, the cells were washed with PBS and incubated with FITC (1:200) or Cy3 (1:400) conjugated secondary antibodies for 1 h at room temperature. The nuclei were counterstained with DAPI (Invitrogen, Barcelona, Spain) and the coverslips were mounted onto glass slides using Aqua-Poly/Mount (Polysciences Europe GmbH, Bergstrasse, Germany). Confocal images were acquired on a TCS SPE microscope from Leica Microsystems (Wetzlar, Germany) and they were analyzed using the ImageJ and Leica LAS AF Lite software.
6
Tracheal Epithelium Quantification Protocol
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Tracheal epithelial extension length was determined in the immunofluorescent images by Ecadherin staining acquired with a Leica TCS SPE microscope with a 10x objective lens (Leica Microsystems) in the middle section of a plate-shaped tracheal section (Figure 1c, dashed line). We measured the tracheal epithelial extension from the edge of the tracheal cartilage prepared before culture. The starting points of the measurement were identified in images (Figures 1d and6c) as the point on the luminal surface of the epithelium crossed by the straight line (dotted lines in Figures 1d and6c). This line is perpendicular to the adventitial surface line (dashed lines in Figures 1d and6c) of the tracheal cartilage and attaches to the cartilage edge. We used ImageJ (National Institutes of Health, Bethesda, MD, USA) for the measurement (Figures 1d and6c, x or y). The sum of the measures on both sides (Figure 1d and 6c, x + y) was defined as the extension length of the tracheal epithelium.
The data from one mouse were the averaged extension lengths from one to four plate-shaped tracheal sections from the mouse.
The data from one mouse were the averaged extension lengths from one to four plate-shaped tracheal sections from the mouse.
7
Quantifying Tracheal Epithelial Cell Dimensions
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In an image acquired with a Leica TCS SPE microscope with a 20x objective lens (Leica Microsystems). The area of extended tracheal epithelium was enclosed with a polygonal line and measured by using area measurement function of ImageJ. A scheme of the average width and thickness, and the average area of an epithleial cell is shown in Figure 1e.
1. The average width of an epithelial cell in the extended tracheal epithelium was calculated by dividing the cross-sectional area of the extended tracheal epithelium by the number of DAPI-positive cells in the extended tracheal epithelium.
2. The average thickness of the extended epithelium was calculated by dividing the crosssectional area of the extended tracheal epithelium by the extension length of the tracheal epithelium.
3. The average area of an epithelial cell in the extended tracheal epithelium was calculated by dividing the cross-sectional area of the extended tracheal epithelium by the number of DAPI-positive cells in the extended tracheal epithelium.
1. The average width of an epithelial cell in the extended tracheal epithelium was calculated by dividing the cross-sectional area of the extended tracheal epithelium by the number of DAPI-positive cells in the extended tracheal epithelium.
2. The average thickness of the extended epithelium was calculated by dividing the crosssectional area of the extended tracheal epithelium by the extension length of the tracheal epithelium.
3. The average area of an epithelial cell in the extended tracheal epithelium was calculated by dividing the cross-sectional area of the extended tracheal epithelium by the number of DAPI-positive cells in the extended tracheal epithelium.
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